The present invention relates to generally to genetic engineering of algal and heterokont cells and more specifically to producing recombinant algal cells using bacterial conjugation.
Bacterial conjugation is a process by which genetic material is transferred from donor cells to recipient cells. The transfer of these genes requires complex protein machinery that ensures DNA mobilization and mating pair formation. Conjugation in Gram-negative bacteria is mediated by the Type IV secretion system (T4SS), a large macromolecular complex involved in substrate transport and pilus biogenesis. T4SSs are implicated not only in bacterial conjugation, but also in the secretion of virulence factors to eukaryotic cells. Many effectors secreted by T4SS are virulence factors involved in pathogenic diseases, such as brucellosis, whooping cough, cat scratch disease, pneumonia, or gastric ulcer, caused by bacterial infection with Brucella suis, Bordella pertussis, Bartonella henselea, Legionella pneumonia or Helicobacter pylori, respectively. Further, bacterial conjugation is one of the main mechanisms whereby bacteria become resistant to antibiotics.
Genes encoding the transfer function (including mobilization (MOB) genes and mating pair formation (MPF) genes can be present on an autonomous replicating plasmid (the “conjugative plasmid” or “mobilization plasmid”) or may be integrated into the genome of a bacterial transfer host. These genes that enable transfer of DNA from the host are found in two separate tra gene clusters in E. coli. Genes to be transferred into the recipient cell can be present on a conjugative plasmid that includes the genes encoding the DNA transfer functions, or can be present on a separate episome that may be referred to as a “DNA transfer construct”, “transfer construct” or “transfer plasmid” (sometimes referred to as a “cargo plasmid”). The transfer construct/cargo plasmid includes, in addition to one or more genes to be transferred into a recipient cell, an origin of transfer (oriT) that includes a sequence recognized by a “relaxase” that is encoded by a mobilization gene.
There are many examples of bacterial conjugation documented in the art. Christie et al. reviews T4SS conjugation systems from data derived from a variety of bacteria, including Agrobacterium tumefaciens, Bordetella pertussis, Helicobacter pylori and Legionella pneumonia (Christie Mol Microbiol (2001) 40(2):294). Schroder et al. showed that type IV secretion system (T4SS) dependent DNA transfer into host cells may occur naturally during human infection with Bartonella (Schroder et al. PNAS (2011) 108(35):14643). Cabezon et al. provide a detailed review of bacterial conjugation and the proteins involved (Cabezon et al. FEMS Microbiol Reviews (2015) 39:81). In a review, Lawley et al. describe that F factor conjugation is a true T4SS in E. coli (Lawley et al. FEMS Microbiol Letters (2003) 224:1). Stucken et al. describe the transformation of the (prokaryotic) cyanobacterial species Fischerella and Chlorogleopsis by conjugation, electroporation and biolostic DNA transfer methods (Stucken et al. Curr Microbiol (2013) 65:552). Anand et al. describe genetic transformation of plants by the bacterium Agrobacterium tumefaciens, which is a plant parasite (Anand et al. New Phytologist (2012) 195:203). Fitzpatrick reviews conjugation in fungi (Fitzpatrick FEMS Microbiol Letters (2012) 329:1). Conjugation resulting in transfer of DNA from a bacterium into a diatom has not been described, and conjugation from a non-Rhizhobial bacterium into a eukaryotic microalga has also not been described.
Diatoms are eukaryotic phytoplankton that contribute a significant fraction of global primary productivity and demonstrate great potential for autotrophic bioproduction of fuels and higher value chemicals. A unique feature of diatoms (which are classified as microalgae and are also heterokonts) is that they are encased in a silca cell wall called a frustule. Although methods for genetic manipulation currently exist for some diatom species, they are slow compared to the efficient methods available for other model microbes such as E. coli and yeast, and this has stymied both basic diatom research and applied strain development. To accelerate research in ecologically and biotechnologically important microalgae, improved transformation methods and episomal vectors were developed.
Episomes can provide a reliable, consistent and predictable platform for protein expression by avoiding the complications of random chromosomal integration that include multiple insertions, position-specific effects on expression, and potential knock-out of non-targeted genes. Consistent protein expression from episomes can also allow for efficient complementation of mutants to confirm gene function. Circular DNA molecules have been previously isolated from diatoms, but they have never been successfully reintroduced as episomes.
Nucleic acid molecules such as episomes can be efficiently moved among bacteria and even between bacteria and eukaryotes via conjugation. To date, cells such as plant and algae cells that have highly structured cell walls have been reported to be transformed by conjugation only by Agrobacterium species (of the family Rhizobiaceae) which are naturally pathogenic to plants. During infection of plant cells by an Agrobacterium species, DNA is transferred from an Agrobacterium donor cell and is integrated into the recipient host genome.